The broad, long-term objective is to elucidate the mechanisms for recovery of the dark state through increase in cytoplasmic cGMP concentration in vertebrate retina rod outer segments (ROS). Biochemical and electrophysiological studies have shown that cGMP plays a key role in the system that converts a light signal into electric signa's in ROS. Little is known about the regulatory mechanism of cGMP synthesis by retinal guanylate cyclase (retGC) in ROS. Other mechanisms for the increase of free cGMP concentration have never been revealed. This proposal is based upon our recent progress in the basic characterization of vertebrate retGC, finding of possible multiple signaling pathways in ROS, and elucidation of unexpected involvement of cGMP phosphodiesterase (PDE) in increase of free cGMP concentration in ROS. Further characterization of the molecular mechanism for the increase of cytoplasmic cGMP concentration with or without cGMP metabolism will provide new aspects to the mechanism of abnormal regulation of cGMP concentration found in retinal cell degeneration, although the proposed research is based on basic biochemical and molecular biological experiments. The proposed research is also expected to provide broad applicability in studies about cellular regulation of second messengers in other cells. Specific Aims in the proposals are: l. To characterize retGC in vertebrate ROS. We have purified retGC from vertebrate ROS and provided basic characterization of retGC. During our study, we found that retGC has special characteristics which are different from other GCs. We also suggested unexpected localization of retGC. Polymorphism of retGC, and its post-translational modifications which may result in the polymorphism of retGC, have also been suggested. The molecular bases and biological significance of these retGC properties will be investigated using biochemical, molecular biological and histochemical methods. 2. To elucidate regulatory mechanisms of retGC in vertebrate ROS. We suggested complex regulatory mechanism of retGC in ROS. Molecular mechanisms and physiological significance of these regulations remain basically unclear. Exploration of new retGC regulatory mechanisms is possible by our progress in preparation of various retGC samples, and purification of Ca binding proteins and transducin isoforms. 3. To explore the direct, positive involvement of PDE in the recovery process of ROS to the dark condition. We recently found the Ca-sensitive cGMP release from PDE by GTP/transducin, retinal G-protein, complex. The unexpected finding allows us to investigate the mechanism of increase of cGMP concentration without cGMP metabolism. These phenomena suggest that the components involved in the reduction of cGMP level may also be involved in the increase of cGMP level in ROS. Molecular mechanism of the regulation will be studied using biochemical and molecular biological methods.